Respiratory Chain Complexes in Dynamic Mitochondria Display a Patchy Distribution in Life Cells

Background

Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism.

Methodology/Principal Findings

The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes.

Conclusion/Significance

Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.     

Disconnecting Mitochondrial Content from Respiratory Chain Capacity in PGC-1-Deficient Skeletal Muscle

1. Download : Download full-size image

     

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp_ha and Fp_hf and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp_hf is reduced more slowly than Fp_ha by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp_m. The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp_1f. In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.     

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.     

Redox State and Mitochondrial Respiratory Chain Function in Skeletal Muscle of LGMD2A Patients

Background

Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.

Methods

We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.

Results

Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls.

Conclusions

Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.     

Identification of New Dystroglycan Complexes in Skeletal Muscle

The dystroglycan complex contains the transmembrane protein β-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.     

Respiratory Chain of Plant Mitochondria: XVIII. Point of Interaction of the Alternate Oxidase with the Respiratory Chain

Oxidation of the respiratory chain carriers of anaerobic, CO-saturated skunk cabbage (Symplocarpus foetidus) mitochondria, by means of an O₂ pulse, proceeds primarily through the cyanide-insensitive alternate oxidase, since the oxidation of cytochromes a and a₃ takes place with a half-time of 3 seconds, corresponding to the rate of dissociation of CO from reduced cytochrome a₃. Ubiquinone and part of the flavoprotein are oxidized within 1 second under these conditions, and this rapid rate of oxidation is strongly inhibited by m-chlorobenzhydroxamic acid (mCLAM), a specific inhibitor of the alternate oxidase of plant mitochondria. The rate of ubiquinone oxidation under these conditions in white potato (Solanum tuberosum) mitochondria, which have no alternate oxidase, is the same as that in skunk cabbage mitochondria treated with mCLAM. Ubiquinone, thus identified as the carrier common to both the cytochrome and alternate oxidase pathways, is linked to the alternate oxidase by a flavoprotein of midpoint potential 50 millivolts more negative with which it is in equilibrium. This arrangement provides a switch for diverting electron transport primarily through the cytochrome pathway under state 3 conditions and primarily through the alternate oxidase pathway under state 4 conditions.     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

Measurement of the Impedance of Frog Skeletal Muscle Fibers

Impedance measurements are necessary to determine the passive electrical properties of cells including the equivalent circuits of the several pathways for current flow. Such measurements are usually made with microelectrodes of high impedance (some 15 MΩ) over a wide frequency range (1-10,000 Hz) and so are subject to many errors. An input amplifier has been developed which has negligible phase shift in this frequency range because it uses negative feedback to keep tiny the voltage on top of the microelectrode. An important source of artifact is the extracellular potential produced by capacitive current flow through the wall of the microelectrodes and the effective resistance of the bathing solution. This artifact is reduced some 10 times by shielding the current microelectrode with a conductive paint. The residual artifact is analyzed, measured, and subtracted from our results. The interelectrode coupling capacitance is reduced below 2 × 10^-17 F and can be neglected. Phase and amplitude measurements are made with phase-sensitive detectors insensitive to noise. The entire apparatus is calibrated at different signal to noise ratios and the nature of the extracellular potential is investigated. The phase shift in the last 5-20 μm of the microelectrode tip is shown to be small and quite independent of frequency under several conditions. Experimental measurements of the phase characteristic of muscle fibers in normal Ringer are presented. The improvements in apparatus and the physiological significance of impedance measurements are discussed. It is suggested that the interpretation of impedance measurements is sensitive to small errors and so it is necessary to present objective evidence of the reliability of one's apparatus and measurements.     

Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle

Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.     

The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain

The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa₃ and c₅₄₇ have unaltered oxidation half-times, designated t_{½ on}, as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t_{½ off}, decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t_{½ on}, for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t_{½ off} decreases only 30% while the extent of oxidation decreases 50%. The values of t_{½ on} and t_{½ off} and the extent of oxidation of cytochrome b₅₅₃ and flavoprotein Fp_ha were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

Fasting Decreases the Content of D-Chiroinositol in Human Skeletal Muscle

Two classes of inositol phosphoglycans have been implicated as second messengers of insulin, one that activates pyruvate dehydrogenase and contains D-chiroinositol, and one that inhibits cyclic AMP–dependent protein kinase and contains myoinositol. We examined the effects of a 3-day fast on muscle contents of inositols in healthy humans. An oral glucose tolerance test was performed and a biopsy was obtained from the quadriceps femoris muscle after an overnight fast and after a 72-hour fast. The 72-hour fast significantly increased plasma glucose (1.5- to 2-fold) and insulin (2- to 4-fold) after glucose ingestion versus the values after the overnight fast, indicating the manifestation of peripheral insulin resistance. The 72-hour fast resulted in an ∼20% decrease in the muscle content of D-chiroinositol (P < 0.02), but no change in the myoinositol content. These data demonstrate that fasting specifically decreases the muscle content of D-chiroinositol in human muscle and this may contribute to the finding that insulin-mediated activation of pyruvate dehydrogenase is attenuated after short-term starvation.     

疲劳性运动中线粒体电子漏引起质子漏增加

以大鼠递增强度力竭性竭性跑台运动为疲劳运动模型,观察了运动后大鼠骨骼肌线粒体电子漏和质子漏的变化。结果表明,运动性疲劳状态下大鼠骨骼肌线粒体超氧阴离子生成增加,脂质过氧线粒体质子漏增多是氧化磷酸化偶联程度下降的重要因素。实验结果支持电子漏引起质子漏的假说。     

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria

The oxidation kinetics of the two high potential flavo-proteins, one (Fp_hf) fluorescent and the other (Fp_ha) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp_ha which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp_ha about 3-fold, but it has no effect on the oxidation half-time of Fp_hf. In contrast to these two inhibitors, m-chlorobenzhydroxamic acid—an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria—increases the oxidation half-time of Fp_hf 10-fold to about 2 seconds, while increasing that of Fp_ha only some 20%. This result implies that the flavoprotein Fp_hf mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp_ha, ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b₅₅₇ is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b₅₅₇ plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b₇ found in mitochondria from the spadices of Arum maculatum.     

Genomic Organization of the Human Skeletal Muscle Sodium Channel Gene

Voltage-dependent sodium channels are essential for normal membrane excitability and contractility in adult skeletal muscle. The gene encoding the principal sodium channel α-subunit isoform in human skeletal muscle (SCN4A) has recently been shown to harbor point mutations in certain hereditary forms of periodic paralysis. We have carried out an analysis of the detailed structure of this gene including delineation of intron-exon boundaries by genomic DNA cloning and sequence analysis. The complete coding region of SCN4A is found in 32.5 kb of genomic DNA and consists of 24 exons (54 to > 2.2 kb) and 23 introns (97 bp-4.85 kb). The exon organization of the gene shows no relationship to the predicted functional domains of the channel protein and splice junctions interrupt many of the transmembrane segments. The genomic organization of sodium channels may have been partially conserved during evolution as evidenced by the observation that 10 of the 24 splice junctions in SCN4A are positioned in homologous locations in a putative sodium channel gene in Drosophila (para). The information presented here should be extremely useful both for further identifying sodium channel mutations and for gaining a better understanding of sodium channel evolution.